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A rapid and reliable protocol was developed to produce chemical degradation of the RNA in vitro. Cell line MDA-MB-453 was used as source of RNA and hydrolysis in water at 70°C was performed. The outcome was monitored by RT-qPCR of two housekeeping targets and a significant correlation between the Ct and the extent of molecular damage of the template was found. A molecular degradation index of the RNA is proposed.

A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6) s(-1) for adenine and 6.27×10(-6) s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing.

Hereby we describe a pilot study for the rational design and synthesis of a glycoconjugate vaccine against Tuberculosis (TB) by site-specific coupling of well-defined glycans to non-antigenic amino acids in a selected protein carrier. A combination of ESI-MS and LC-MS analytical methods was applied for the systematic characterization of the reactivity of the surface amino acids in the glycosylation reaction with monosaccharides towards 2-iminomethoxyethyl or homobifunctional (4-nitrophenyl ester) linkers, both on the model protein, ribonuclease A (RNase A) and on TB10.4, the simplest antigenic protein isolated from Mycobacterium tuberculosis (MTB). Intact protein analysis was carried out to quantify the glycosylation degree and profile the glycoform composition of all the prepared neo glycoconjugates, while pronase and chymotriptic digests were analyzed to map and rank the reactivity of protein residues. Neo glycopeptides were purified by on-line porous graphitized carbon solid-phase extraction, separated by hydrophilic interaction liquid chromatography and analyzed by electrospray mass spectrometry (ESI-MS(n)). Significantly, different site specificity and glycosylation efficiency were demonstrated for the two linkers, resulting in structurally diverse glycoconjugates. A computational analysis of the amino acids involved in the epitope formation in TB10.4 addressed the choice to 2-iminomethoxyethyl-saccharide activation, that resulted in a more targeted and selective conjugation preserving the protein antigenicity. Additionally, a rational design of experiments lead to the identification of suitable experimental conditions for the preparation of highly pure and homogeneous neo glycoconjugates.

A capillary electrophoresis (CE) method was used to study the DNA primary structure of artificially degraded DNA samples and aged/forensic specimens. CE investigated the ratio between the pyrimidinic and the purinic canonical moieties still bound on the DNA backbone and identified the presence of additional compound (i.e. un-canonical DNA bases). The DNA samples were then analysed by using molecular assays such as agarose gel, UV spectrophotometry and qPCR. All these data have been concurrently considered for a critical evaluation of the outcome of the STR typing.